Recent studies indicate that diabetes is associated with an increased incidence of respiratory failure, a heightened risk of postoperative respiratory complications, and a greater need for prolonged mechanical ventilation. However, it is not known how diabetes produces these problems. One potential explanation is that uncontrolled diabetes alters respiratory muscle function, reducing the capacity of the respiratory pump. The purpose of the present proposal is to examine this issue. Our central hypothesis is that poorly controlled diabetes induces severe free radical mediated diaphragm dysfunction. We will test this hypothesis in the following groups of studies. Aim I studies will characterize the effects of uncontrolled diabetes on diaphragm specific force generation, changes in muscle mass, and diaphragm endurance, testing the hypothesis diabetes induced alterations in diaphragm performance are related to increases in free radical generation. Aim II studies will interrogate a number of free radical generating pathways in muscle (including the cell surface NADPH oxidase) and determine which pathways are responsible for increased free radical generation in the diaphragm in diabetes. Aim III studies will determine if iNOS is upregulated in the diaphragm in diabetes, and will test the hypothesis that iNOS acts as an upstream modulator of free radical generation. Aim IV studies will examine several downstream targets of diabetes induced free radical generation in the diaphragm that are responsible for reductions in diaphragm performance, including contractile protein alterations, calpain mediated reductions in muscle mass, and alterations in mitochondrial ATP generating capacity. A variety of physiologic, biochemical, proteomic, fluorogenic, pharmacologic and genetic techniques will be used to test these hypotheses. Our preliminary data represent the first demonstration of upregulation of NADPH oxidase subunit proteins in skeletal muscle in any disease process, and suggest that iNOS regulates NADPH oxidase activity and free radical generation in the diaphragm in diabetes. These new data should provide important information regarding the pathogenesis of diabetes induced diaphragm dysfunction, and uncover pathways which could provide novel therapeutic targets for treatment of respiratory muscle weakness in this condition.